Hybrid phosphoinositide phospholipids: compositions and uses

ABSTRACT

The methods and compositions disclosed herein concern the synthesis of a novel class of “two-headed” phospholipid-phosphoinositide hybrids possessing a carbon backbone, such as 2,3-diacylthreitol, erythritol or a synthetic module. The second phospholipid head group allows introduction of a biochemical or chemical moiety in a position orthogonal in space to those occupied by the phosphoinositide head group and the two acyl chains. The diacyl moieties allow for the incorporation of Pea-PIP 2  into a lipid bilayer, while the Ptdlns(4,5)P 2  moiety in the aqueous layer is specifically recognized by lipid binding proteins. In alternative embodiments of the invention, reporters, for example biotin, fluorophores and/or spin labels, are attached to the free amino group of the head groups of such molecules to specifically target the reporters to the lipid-water interface.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 60/368,556 filed Mar. 29, 2002 and 60/392,783 filed Jun.28, 2002.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

The U.S. Government has certain rights in this invention based uponpartial support by National Institutes of Health Grant Number NS-29632.

BACKGROUND OF THE INVENTION

Phosphoinositides (“PtdlnsP_(n)s”) are biosynthesized by the interplayof kinases and phosphatases. These charged lipids are minor componentsof cellular membranes but are vital as second messengers for diversecellular functions. PtdlnsP_(n)s are essential elements in tyrosinekinase, growth factor receptor and G-protein receptor signalingpathways. Furthermore, these lipid signals have important roles inmembrane trafficking, including endocytosis, exocytosis, Golgi vesiclemovement and protein trafficking, in cell adhesion and migration, inremodeling of the actin cytoskeleton, and in mitogenesis andoncogenesis. Activation of cellular signaling pathways often resultsfrom production of one of eight specific PtdlnsP_(n)s in response to astimulus, and each PtdlnsP_(n) has a specific role for a given signalingpathway in each cell-type.

Phosphoinositide recognition by binding proteins and lipid-metabolizingenzymes involves specific interactions with the phosphoinositide headgroup and diacylglycerol backbone which vary significantly from proteinto protein. When a fluorescent probe is introduced into the inositolhead group, binding and metabolism can be attenuated or abrogatedentirely. Moreover, many acyl-modified phosphoinositides fail to showadequate K_(m) and V_(max) values as substrates for lipid kinases andphosphatases. Further, certain phosphoinositide binding proteinsdemonstrate reduced binding to head group- or acyl-modifiedphosphoinositides. The simple, robust assays needed for biochemical andcellular studies require a chemically-modified phosphoinositidesubstrate that can be both acted on by enzymes and recognized byspecific binding proteins. A need exists for novel types ofphosphoinositides derivatives whose modifications are consistent withthe natural binding affinities and sub-cellular localization of thenative compounds. Derivatives of this sort would also function as toolsuseful in drug discovery or drug development assays, and for basicresearch.

SUMMARY OF THE INVENTION

The present methods and compositions relate to the fields ofpharmacology and drug discovery. More particularly, the methods andcompositions concern derivatives of phosphatidylethanolamine-extendedphosphoinositides and phosphoinositide polyphosphates (PtdlnsP_(n)s) anduse thereof in drug discovery and development of assays, as well as forbasic research purposes. The present invention concerns the design andasymmetric total synthesis of the first examples of a new class offunctionalized PtdlnsP_(n)s, the Pea-PIP_(n)s.

The synthetic strategy involves homologation of the 1,2-diacylglycerolbackbone to a carbon threitol backbone, such as 2,3-diacylthreitol,erythritol or synthetic module. As seen in FIG. 1, such hybrid lipidspossess a phosphatidylethanolamine (PE, or Pea) head group at the1-position and a PtdlnsP_(n) head group at the 3,4 and/or 5-position. Areporter group, for example biotin, a fluorophore, or a spin label, maythen be covalently attached to the free Pea amino group. FIG. 1 shows anunmodified dipalmitoyl Ptdlns(4,5)P₂ at center, with the acyl-modifiedNBD (fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) derivative aboveand an exemplary Pea-PIP_(n) NBD derivative at the bottom. The reportergroups in these synthetic constructs are targeted to the lipid-waterinterface at a site distant from the key PtdlnsP_(n) head grouprecognition features. The unchanged diacyl moiety permits insertion andretention of Pea-PIP_(n)s in a lipid bilayer to facilitate recruitmentof PtdlnsP_(n)-specific binding proteins to a membrane surfaceenvironment.

These new hybrid lipids can serve as direct enzymatic substrates thatcan be delivered into a cell in order to measure direct turnover. Thisis a great improvement over measuring competitive displacement with asurrogate. Accordingly, Pea-PIP_(n)s of the present invention havepotential for the production of unique, reporter-based high throughputscreens or assays for in vitro biochemical activity and for monitoringreal time in situ biochemical activity in living cells.

BRIEF DESCRIPTION OF THE FIGURES AND DRAWINGS

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the disclosedembodiments of the invention. The embodiments of the invention may bebetter understood by reference to one or more of the figures incombination with the detailed description of specific embodiments of theinvention presented herein.

FIG. 1. Illustrates (at c) an exemplary hybrid lipid of the presentinvention that possess a phosphatidylethanolamine (PE, or Pea) headgroup at the 1-position and a Ptdlns(4,5)P₂ head group at the 4-positionin accordance with one embodiment of the present invention. Shown are(a), the unmodified Dipalmitoyl-Ptdlns(4,5)P₂ at the center; (b), theacyl-modified NBD-derivative above (1-C₆-NBD,2-C₆-Ptdlns(4,5)P₂); and(c), the Pea-PIP_(n) NBD derivative at the bottom (NBD-Pea-PI(4,5)P₂).

FIG. 2. Illustrates synthesis of exemplary Pea-PIP_(n)s, includingprotection debenzylation by hydrogenolysis resulting in each of eightdesired Pea-PIP_(n)s.

FIG. 3. Illustrates exemplary synthesis of differentially functionalized2,3-diacylthreitol backbones in accordance with an embodiment of thepresent invention. Reagents and conditions: (a) cyclopentanone, pTSA,toluene, reflux; (b) LiAlH₄, THF; (c) NaH, p-methylbenzyl (PMB)Cl, DMF;(d) 1-H-tetrazole, Cbz-aminoethyl phosphoramidite 4, CH₂Cl₂; (e) 1 MHCl, tetrahydrofuran (THF); (f) C₁₅H₃₁COOH, dicyclohexylcarbodiimide(DCC), DMAP, CH₂Cl₂; (9) dichlorodicyanoquinone (DDQ), CH₂Cl₂/H₂O.

FIG. 4. Illustrates exemplary backbone phosphorylation and synthesis ofPea-PIP₂ derivatives 12. Reagents and conditions: (a) BnOP(NiPr₂)₂,1-H-tetrazole, CH₂Cl₂; (b) 1-H-tetrazole, 4,5-HG, CH₂Cl₂; (c) H₂ (60psi), 10% Pd/C, THF/H₂O; (d) probe-NHS ester, 0.5 M TEAB, DMF.

FIG. 5. Illustrates exemplary Pea-PIP_(n) reporter groups according toan embodiment of the present invention. Reaction of the free Pea aminogroup of with four N-hydroxysuccinimidyl (NHS) esters afforded thecorresponding biotinylated derivative 4 a, the fluorescentN-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD) and 6-carboxyfluoresceinderivatives 4-b and 4-c, and the spin-labeled3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PROXYL) derivative 4-d.

FIG. 6. Illustrates an exemplary linker-modified Pea-PIP, analog whereinthe linker modification is an amino-PEG-amide.

FIG. 7. Illustrates the synthesis of exemplary short PEG linkers.

FIG. 8. Illustrates the synthesis of an exemplary protected threitolbackbone with PEG linkers.

DETAILED DESCRIPTION

One embodiment of the present invention comprises a strategy forsynthesizing a novel class of “two-headed” phospholipid-phosphoinositidehybrids possessing a carbon threitol backbone, such as a2,3-diacylthreitol, erythritol or synthetic module. These hybrid lipidspossess a phosphatidylethanolamine (PE or Pea) head group at the1-position and a PtdlnsPn head group, such as Ptdlns(4,5)P₂, at the 3,4and/or 5-position. In particular embodiments, a reporter group, e.g.,biotin, a fluorophore, or a chelating agent, may then be covalentlyattached to the free Pea amino group. The reporter would thus betargeted to the lipid-water interface at a site distant from the keyPtdlnsPn head group (for example, Ptdlns(4,5)P₂) recognition features ofthe binding protein. In various embodiments, the diacyl moiety permitsinsertion and retention of Pea-PIP_(n)s in a lipid bilayer to facilitaterecruitment of PtdlnsPn head group—(for example, Ptdlns(4,5)P₂—)specific binding proteins to a membrane surface environment.

The additional phospholipid head group allows introduction of abiochemical or chemical moiety in a position orthogonal in space tothose occupied by the phosphoinositide (“PIP_(n)”) head group and thetwo acyl chains. The method for producing the hybrid phospholipids ofthe present invention involves diethyl D-tartrate as the chiralprecursor for the extended glycerol backbone of the target hybrid lipid.The corresponding acetal is reducted with lithium aluminum hydride andprotected with 1 equivalent of PMB-Cl to give the monobenzyl ether.Coupling with a carbonylbenloxy (Cbz)-protected aminoethoxyphosphoramidite yields, after oxidation, the protected phosphatidylanalogue ready for addition of the acyl chains and the selected PIP_(n)head group. After acetal hydrolysis, the 2,3-dipalmitoyl derivative isprepared. As shown for exemplary molecules in FIG. 2, the functionalizedcarbon threitol backbone is then coupled with a phosphatidyl head group.Debenzylation by hydrogenolysis then affords, in particular embodiments,each desired phosphatidylethanolamine-phosphoinositide (“Pea-PIP_(n)”)with a free amino groups ready for derivatization.

Any PIP_(n) head group may be prepared and used according to theinvention. PIP_(n) head groups to be utilized in this invention may becommercially obtained or prepared by standard methods known in the art.

In a particular embodiment of the invention, members of this class ofmolecules have a phosphatidylethanolamine (“Pea”) head group at the1-position and a phosphoinositide (“PIP_(n)”) head group at the 3,4and/or 5-position.

According to this embodiment, the Pea-diacylthreitol synthetic module isprepared and coupled with any selected phosphoinositide head group. Forexample, each of eight different naturally occurring phosphoinositidehead groups (PI, PI(3)_(p), PI(4)P, PI(5)P, PI(4,5)P₂, PI(3,4)P₂,PI(3,5)P₂, PI(3,4,5)P₃) has been used to produce Pea-PIP_(n)s of thepresent invention (the head groups used for synthesizing Pea-PIP_(n)sare available commercially, for example from Echelon Biosciences Inc.,SLC Utah). Accordingly, certain embodiments of the present inventioninclude Pea-PI, Pea-PI(3)P, Pea-PI(4)P, Pea-PI(5)P, Pea-PI(3,4)P₂,Pea-PI(3,5)P₂, Pea-PI(4,5)P₂ and Pea-PI(3,4,5)P₃.

In another embodiment of the present invention, a linker may be utilizedbetween the PIP_(n) headgroup and a reporter. In a particularembodiment, the length of the linker between the PIP_(n) headgroup andthe reporter moiety at the primary amine is lengthened. In a particularembodiment, the extension for the linker comprises an oligo-polyethyleneglycol linker. According to this embodiment, a commercially availabledi-, tri-, tetra-, and/or penta(ethylene glycol) is used for extendingthe linker during synthesis of the Pea-PIP_(n)s.

Other embodiments include additional or alternative linkers. Forexample, one such alternative linker is a diamino linker that will yielda phosphoramidate final product rather than a phosphate linkage. Anotheralternative linker uses both phospho- and non-phospho linked spacers. Incertain embodiments, this can be accomplished by replacing the Pea groupat C-4, such as with a simple ester, amide or other linkage that allowsa pendant functionality to be incorporated. Another alternative linkeris to use a phosphatidylserine or other carboxylic acid instead of Peato permit further functionalization at the end distal to the PIP_(n)recognition element Various methods for producing alternative linkersare well known in the art.

Pea extension of the invention may also include aminoalcohols aslinkers, for example 3-aminopropanol, 4-aminobutanol, and others. Incertain embodiments heteroatom-containing derivatives such as1-amino-11-hydroxy-3,6,9-trixaundecane or similar aminoalcohols withwater soluble spacer chains may be used. These can include branchedaminoalcohols including, but not limited to, for example,2-aminomethyl-3-amino-1-propanol which has multiple reactive aminotermini for addition of two or more biochemical probes. One of ordinaryskill in the art will know methods of Pea extension beyond those listedabove.

According to certain embodiments of the present invention, the acylchains to be attached to any of the above mentioned head groups mayinclude any acyl group from n=2 to n=26 carbons. Methods of modifyingthe length or degree of double bonds in an acyl chain are well known inthe art. In certain embodiments, the carbons will have a number ofdouble bonds, for example from 0 to 6. In alternative embodiments, oneor both acyl chains can be replaced with an ether chain of the samelength, degree of unsaturation or terminal functionalization.Replacement of one or both acyl chains can be accomplished by anystandard method known in the art.

According to other embodiments, the phosphate groups of this inventioncan be chemically modified to increase stability or resistance tochemical or enzymatic hydrolysis. In certain embodiments, the phosphategroups will be on the inositol, the phosphodiester or the PEphosphodiester. In alternative embodiments, this involves a P═S or P—Sbond, replacement of a P—O phosphate linkage with a P—CH₂, P—CHF orP—CF₂ phosphonate linkage or a phosphoramidate linkage. Other methods ofchemically modifying the phosphate groups of this invention are known inthe art.

In certain embodiments, a Pea-PIP_(n) of the present invention has atriester analog at P-1 to allow for an additional site for derivation.Methods of synthesizing a Pea-PIP_(n) with a triester analog at P-1 arewell known in the art. (See, e.g., Q.-M. Gu and G. D. Prestwich,“Synthesis of Phosphotriester Analogues of the PhosphoinositidesPtdlns(4,5)P2 and Ptdlns(3,4,5)P3,” J. Org. Chem., 61, 8642-8647(1996)).

In yet another embodiment, the Pea-PIP_(n) carbon threitol backbone caninclude four, five, six or more carbons. In particular embodiments, suchcarbon backbone may be branched. In a certain embodiment, such carbonthreitol backbone is 2,3-diacylthreitol or erythritol.

In yet another embodiment, Pea-PIP_(n)s include a polymerizable groupthat allows for the construction of Pea-PIP_(n)s polymers. Accordingly,particular embodiments of the invention comprise oligomeric Pea-PIP_(n)sformed by linking two or more Pea-PIP_(n) molecules together.

In particular embodiments, a reporter group (or “label” or “tag”) iscovalently attached to the free Pea amino group. Such a reporter mayinclude, for example, a fluorescent label, a radiolabel, achemiluminescent label, a spin label, a photophore, a chromophore,biotin, a nanogold particle, and/or any suitable reporter, and mixturesthereof.

As used herein, suitable fluorescent compounds that can be usedaccording to the present invention include chemically activated,thetherable analogs of acrylodan, AMCA, BODIPY, Cascade-Blue, CINERF,dansyl, dialkylaminocoumarin, eosin, erythrosine, fluorescein (FITC),hydroxycoumarin, NBD, Oregon green, PyMPO, pyrene, rhodamine, RhodolGreen, TMR, Texas Red, X-Rhodamine, and the like.

Attached to the Pea amino group, the reporter would thus be targeted tothe lipid-water interface at a site distant from the specific featuresof the PtdlnsP_(n) head group necessary for interaction with lipidrecognition proteins or other chemicals or compounds that specificallyinteract with the PtdlnsP_(n) at a membrane surface. In variousembodiments, the diacyl moiety permits insertion and retention ofPea-PIP_(n)s in a lipid bilayer to facilitate recruitment ofPtdlnsP_(n)-specific binding proteins to a membrane surface environment.An individual Pea-PIPn may have one or more reporters which may be ofthe same or different types. In various embodiments, the Pea-PIPns willbe transported into cells, with or without a reporter.

All stereoisomers, which include enantiomers or diasteromers, for anycomponent of the Pea-PIP_(n) molecules can be employed in any embodimentof this invention. Such modifications are well known in the art.

Certain embodiments will include Pea-PIP_(n)s bound to a surface, forexample for use in a biochemical assay. In particular embodiments, suchsurface will include a plate, a bead or nitrocellulose. In a particularembodiment, such surfaces are selected from the group consisting of, butnot limited to, a chemically activated glass, plastic or other surface;activated agarose, polystyrene or any other type of bead andnitrocellulose. In other embodiments, the Pea-PIP_(n)s are attached to ametal surface, such as gold. In particular embodiments, attachment togold is accomplished by introducing to the Pea-PIP_(n) a pendant alkylthiol moiety that is capable of attaching to a gold surface. Methods formaking such pendant alkyl thiol derivatives are well known in the art.

In alternative embodiments, the Pea-PIP_(n)s will be incorporated in aliposome. Such liposome incorporated Pea-PIP_(n)s may include areporter.

Certain embodiments allow for the use of Pea-PIP_(n)s for assays. Thecompositions and methods of their use can be used in any assay thatcurrently uses a modified Pea-PIP. Particular embodiments include invitro fluorogenic, FRET, ELISA and chemiluminsence assays. These may bein a high-throughput format. Alternative embodiments include in vitroenzyme assays, lipid kinase or phosphatase activity, cell-based assaysand agonist or antagonist assays.

Such assays include, but are not limited to, in vitro enzyme assays, invitro agonist or antagonist assays or cell-based assays. In someembodiments of the invention, labeled Pea-PIP_(n)s can also be linked orbound to plates, beads or other surfaces which may, in particularembodiments, be coated with a means for binding Pea-PIP_(n) thereto.Labels of use in the present invention may be activated by any methodknown in the art in order to effect attachment to a Pea-PIP_(n) of thepresent invention. Methods of use in the attachment of a label include,but are not limited to, the activation of an ester, carbonyldiimidazoleactivation and use of any Michael acceptor, such as acrylates,acrylamide, maleimides, vinylsulfone, a,b-unsaturated ketones, esters,aldhydes, amides, and the like.

An example of such a means of linking or binding a Pea-PIP_(n)s isstreptavidin. Another example is NHS activation. In other embodiments,the Pea-PIPn is bound to nitrocellulose. In particular embodiments, thereporter is biotin or a fluorescent label. Fluorescent compoundssuitable for use as a label or reporter according to the presentinvention include, but are not limited to, chemically activatedtetherable analogs of acrylodan, AMCA, BODIPY, Cascade-Blue, CINERF,dansyl, dialkylaminocoumarin, eosin, erythosine, fluorescein (FITC),hydroxycoumarin, NBD, Oregon green, PyMPO, pyrene, rhodamine, RhodolGreen, TMR, Texas Red, X-Rhodamine, and the like.

One embodiment of the present invention provides a method of screeningfor phosphoinositide-specific binding proteins in a membrane surfaceenvironment.

In particular embodiments a high throughput screen (HTS) comprisingcompositions of the invention can be used for identifying, for example,chemicals, natural products and/or or synthetic compounds that affectphosphoinositide recognition and/or signaling at a cell membrane. Suchcompounds include, but are not limited to, for example, agonists andantagonists for protein kinases and phosphoinositide kinases and forphosphoinositide and inositol phosphate binding proteins that areregulated by PIP_(n)s or IP_(n)s and may serve as downstream effectorsin signaling pathways important for therapeutic interventions. Inparticular embodiments, lipid phosphatases or phospholipases areidentified. In alternative embodiments, HTS assays include cell-basedassays using intracellular PIP_(n)s introduced by the shuffling systemand can use primary cells, immortalized cells, cancer cells, cellstransformed with plasmids encoding key enzymes or other proteins, andthe like. The assays could also use in vitro cell extracts or partiallypurified or homogeneous proteins.

In other embodiments the Pea-PIP_(n)s are introduced into cells. Suchintroduced Pea-PIP_(n)s may be labeled, or tagged, with one or morereporters. In a particular embodiment, fluorescent acyl-modifiedPea-PIP_(n)s are shuttled into cells where they exhibit subsequentappropriate membrane localization.

In certain embodiments, assays according to the present invention areperformed in living cells. Particular embodiments of the inventionprovide compositions and methods for visualizing the location of labeledphosphoinositides within a cell. A method for facilitating uptake of aPea-PIP_(n)s into a cell comprises contacting the cell with acomposition of matter comprising a Pea-PIP_(n) and a shuttle, or othermethod of introducing the Pea-PIP_(n) into the cell. Compounds suitablefor shuttling Pea-PIP_(n)s into a cell can include, but are not limitedto, polyamines. Such polyamines can include, for example,aminoglycosidic aminocyclitols (e.g., aminoglycoside antibiotics),synthetic “spherical” dendrimeric polyamines, polybasic nuclear proteins(histones), polybasic polypeptides, lipidic polyamines,polyethyleneimine, steroidal polyamines, and the like, and mixturesthereof. Other polybasic proteins (or polybasic polypeptides) useful forintroducing a Pea-PIP_(n) into a cell include proteins or polypeptidethat contains sufficient lysine, arginine, and/or histidine residues tocomplex an anionic ligand, such as an Pea-PIP_(n). The polybasicpolypeptide may also contain unnatural or non-protein amino acids,N-acylglycine groups, and any of a known group of amide groupreplacements known as peptide bond isosteres. In particular embodiments,assays performed in living cells are monitored by high-content screeningmethods or confocal microscopy. Such in vitro assays allow for minimaldisruption of the normal cellular environment

The disclosed compositions and methods of their use can be used for thediscovery of new pharmaceutical agents and targets related toPtdlnsP_(n)s compounds.

The skilled artisan will realize that the chemical modifications listedabove are exemplary only and that many variations may be used, dependingon the particular type of Pea-PIP, to be synthesized.

Definitions

For the purposes of the present invention, the following terms shallhave the following meanings:

“Tag” or “labels” are used interchangeably to refer to any atom,molecule, compound or composition that can be used to identify aPea-PIP, to which the label is attached. In various embodiments of theinvention, such attachment may be either covalent or non-covalent Incertain embodiments of the invention, the labels have physicalcharacteristics that facilitate the identification of the label. Innon-limiting examples, labels may be fluorescent, phosphorescent,luminescent, electroluminescent, chemiluminescent or any bulky group ormay exhibit Raman or other spectroscopic characteristics. It isanticipated that virtually any technique capable of detecting andidentifying a labeled nucleotide may be used, including visible light,ultraviolet and infrared spectroscopy, Raman spectroscopy, nuclearmagnetic resonance, electron paramagnetic resonance, positron emissiontomography, scanning probe microscopy and other methods known in theart.

Moreover, for the purposes of the present invention, “a” or “an” entityrefers to one or more of that entity; for example, “a Pea-PIP” or “aPea-PIP_(n)” refers to one or more of the compound or at least onecompound. As such, the terms “a” or “an”, “one or more” and “at leastone” can be used interchangeably herein. It is also noted that the terms“comprising,” “including,” and “having” can be used interchangeably.

Furthermore, a compound “selected from the group consisting of” refersto one or more of the compounds in the list that follows, includingmixtures (i.e. combinations) of two or more of the compounds.

According to the present invention, an isolated or biologically puremolecule is a compound that has been removed from its natural milieu. Assuch, “isolated” and “biologically pure” do not necessarily reflect theextent to which the compound has been purified. An isolated compound ofthe present invention can be obtained from its natural source, can beproduced using laboratory synthetic techniques or can be produced by anysuch chemical synthetic route.

As used herein, “shuttle” means a compound, polymer, complex, or mixturethereof that facilitates transport of phosphoinositides, inositolpolyphosphates, and mixtures thereof into cells. Preferred shuttlescomprise polyamines.

EXAMPLES

It should be appreciated by those skilled in the art that the techniquesdisclosed in the examples which follow represent techniques discoveredby the inventors to function well in the practice of the invention, andthus can be considered to constitute the preferred modes for itspractice. However, those of skill in the art should appreciate, in lightof the present disclosure, that many changes can be made in the specificembodiments disclosed herein which will still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Synthesis of Functionalized Phosphoinositide Polyphosphates,the Pea-PIP_(n)s, and Reporter Analogs

The general method for synthesis of Pea-PIP_(n)s of the presentinvention is described in Rzepecki, P. W. and Prestwich, G. D.: J. Org.Chem. 2002, 67(16): 5454-60, which is hereby incorporated by referencein its entirety. In the steps disclosed below, the numbers in boldrefer- to compounds and synthetic intermediates shown in FIGS. 3, 4 and5.

Diethyl D-tartrate was chosen as the chiral precursor for the extendedglycerol backbone of the target hybrid lipid. The absolute configurationof both stereogenic centers at C-2 and C-3 is identical to theconfiguration of glycerol sn-2 position in naturally-occurringPtdlnsP_(n)s and in natural Pea. Moreover, the C₂ axis allowed the useof a monoprotection step in the early stages of the synthesis. Synthesisof an exemplary embodiment, Pea-PI(4,5)P₂ and reporter analogs is shownin FIGS. 3, 4 and 5. Diethyl D-tartrate 1 was protected as acyclopentylidene acetal, which was found to be most readily removedafter backbone functionalization. Initially, an isopropylidene acetalwas used, but scale-up of the deprotection led to unsatisfactory yieldsof the desired intermediate. Reduction of acetal 2 with lithium aluminumhydride afforded (2R,3R)-O-cyclopentylidene threitol 3 a, which wasprotected with 1 equiv. of PMB-Cl to give the monobenzyl ether 3 b. Theprimary alcohol of 3 b was converted to a Cbz-protected Pea head groupby coupling to the phosphoramidite 4, which after oxidation afforded theprotected PE analogue 5. Acidic hydrolysis yielded diol 6 in 65% yieldafter silica chromatography. Acylation with palmitic acid provideddiester 7, and oxidative cleavage of the p-methoxybenzyl (PMB) withdichlorodicyanoquinone (DDQ) gave primary alcohol 8.

FIG. 4 illustrates the installation of two different phosphorylated headgroups on the 2,3-diacylthreitol backbone. Thus, reaction of alcohol 8with benzyltetraisopropylphosphordiamidite yielded a homologatedPea-like phosphoramidite reagent 9, which was coupled with the protectedmyoinositol 4,5-bisphosphate head group obtained as previously described(Prestwich, G. D.; Chaudhary, A; Chen, J.; Feng, L; B. Mehrotra; Peng,J. In Phosphoinositides: Chemistry, Biochemistry and BiomedicalApplications; Bruzik, K. S., Ed.; American Chemical Society: Washington,D.C., 1999; Vol. 818, p 24-37), to give the fully protected Pea-PIP₂precursor 10. Global debenzylation of 10 was accomplished byhydrogenolysis to give the free phosphate monoesters and phosphodiestersin the hybrid lipid Pea-PIP₂ (11). Reaction of the free amino group of11 with four succinimidyl esters afforded the corresponding biotinylatedderivative 12 a, the fluorescent NBD and 6-carboxyfluoresceinderivatives 12 b and 12 c, and the spin-labeled PROXYL(tetramethyl-1-pyrrolidinyloxy) derivative 12 d. Biological results aredescribed below for biotinylated derivative 12 a and fluorescentanalogue 12 c. The spin-labeled derivative 12 d may be used to probeinterfacial protein-lipid interactions in liposomes, in analogy to theuse of acyl spin-labeled probes to characterize the MARCKSpeptide-Ptdlns(4,5)P₂ interaction in liposomes.

The steps comprising an exemplary synthetic process resulting in thenovel hybrid lipid, Pea-PI(4,5)P₂, are described in detail as follows:

(2R,3R)-1,4-Dioxa-spiro[4.4]nonane-2,3-dicarboxylic acid diethyl ester2. Diethyl D-tartrate 1 (1.004 g, 4.87 mmol), cyclopentanone (2.2 mL,24.35 mmol, 5 equiv.) and p-toluenesulfonic acid (93 mg, 0.49 mmol, 0.1equiv.) were dissolved in toluene (75 mL) and stirred under reflux for36 h with azeotropic removal of water using a Dean-Stark trap. Uponcompletion, the reaction mixture was cooled to room temperature (rt) andneutralized with solid NaHCO₃. Solid salts were filtered off, thefiltrate was concentrated in vacuo, and the crude product was purifiedon SiO₂ (hexane:acetone 4:1 containing 10% v/v Et₃N) to give 1.166 g(4.28 mmol, 88%) of acetal 2 as a colorless oil. ¹H NMR (400 MHz, CDCl₃)□ 4.73 (s, 2H), 4.28 (q, 4H, J=5.4), 1.94-2.04 (m, 2H), 1.80-1.90 (m,2H), 1.65-1.76 (m, 4H), 1.32 (t, 6H, J=5.4); ¹³C NMR (100 MHz, CDCl₃) δ169.67, 123.34, 77.08, 61.85, 36.66, 23.50, 14.15; IR: 2980, 1756, 1337,1119, 1115, 1023, 460, 453. Anal. Calcd for C₁₃H₂₀O₆: C, 57.34; H, 7.40.Found: C, 57.34; H, 7.21.

(2R,3R)-(3-Hydroxymethyl-1,4-dioxa-spiro[4.4]non-2-yl)-methanol 3 a. Asolution of diethyl ester 2 (1165 mg, 4.28 mmol, 1 equiv.) in drytetrahydrofuran (THF) was transferred via canula to a suspension ofLiAlH₄ (244 mg, 6.42 mmol, 1.5 equiv.) in dry THF, pre-cooled inbrine/ice bath. The reaction mixture was stirred at rt for 24 h and thensaturated aq. potassium sodium tartrate was added dropwise to decomposeexcess hydride reagent The mixture was stirred for additional 24 h, andthen extracted with three portions of CH₂Cl₂. The combined organicphases were dried over MgSO₄, concentrated in vacuo, and the crudeproduct was purified on SiO₂ (hexane:acetone 3:2 containing 10% v/vEt₃N) to give 805 mg (4.27 mmol, 99%) of the diol 3 a as a colorlessoil. ¹H NMR (400 MHz, CDCl₃) δ 3.85-3.95 (m, 2H), 3.60-3.80 (m, 4H),1.73-1.85 (m, 4H), 1.60-1.73 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 119.36,78.40, 62.46, 37.32, 23.44; IR: 3390, 2956, 2875, 1434, 1335, 1204,1112, 1041, 973. Anal. Calcd for C₉H₁₆O₄: C, 57.43; H, 8.57. Found: C,57.48; H, 8.52.

(2R,3R)-[344-Methoxy-benzyloxymethyl)-1,4-dioxa-spiro[4.4]non-2-yl-methanol3 b. To a suspension of NaH (175 mg, 4.38 mmol, 1 equiv.) in dry DMF wasadded alcohol 3 a (824 mg, 4.38 mmol, 1 equiv.) in dry DMF via canula.The mixture was cooled to 0° C. and then 4-methoxybenzyl chloride (0.65ml, 4.82 mmol, 1.1 equiv.) was added dropwise over 20 min. The ice bathwas removed and the mixture was stirred at rt for 18 h. Traces of NaHwere decomposed by slow addition of water. The mixture was extracted 3times with CH₂Cl₂, dried (MgSO₄), and concentrated in vacuo. The crudeproduct was purified on SiO₂ (hexane:acetone 4:1 containing 10% v/vEt₃N) to give 879 mg (2.85 mmol, 65%) of compound 3 a as a colorlessoil. ¹H NMR (400 MHz, CDCl₃) δ 7.20-7.30 (m, 2H), 6.80-6.95 (m, 2H),4.45-4.55 (m, 2H), 3.92-4.00 (m, 1H), 3.82-3.89 (m, 1H), 3.79 (s, 3H),3.60-3.76 (m, 3H), 3.49 (dd, 1H. J₁=4.7, J₂=7.4), 2.45 (bs, 1H),1.73-1.89 (m, 4H), 1.58-1.73 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 159.29,129.70, 129.40, 128.52, 119.31, 113.82, 79.56, 76.58, 73.21, 70.14,62.63, 55.19, 37.26, 37.20, 23.52, 23.40. IR: 3466, 2955, 2872, 1612,1514, 1248, 1101, 1035. Anal. Calcd for C₁₇H₂₄O₅: C, 66.21; H, 7.84;Found: C, 66.27, H, 7.76.

[2-(Benzyloxy-diisopropylamino-phosphanyloxy)-ethyl]-carbamic acidbenzyl ester 4. To a solution ofbenzyloxybis(N,N-diisopropylamino)phosphine (1.538 g, 4.54 mmol, 1.5equiv.) and 1-H-tetrazole (106 mg, 1.51 mmol, 0.5 equiv.) in dry CH₂Cl₂,was added a solution of (2-hydroxyethyl)carbamic acid benzyl ester (591mg, 3.03 mmol) in CH₂Cl₂. The mixture was stirred at rt for 3 h,concentrated in vacuo, and the crude product was purified on SiO₂(hexane:acetone:Et₃N 6:4:1) to give 886 mg (2.05 mmol, 68%) of compound4 as an air- and moisture-sensitive colorless oil. ¹H NMR: (400 MHz,CDCl₃) δ 7.20-7.40 (m, 10H), 5.21 (bs, 1H), 5.08 (s, 2H), 4.69 (m, 2H),3.64-3.79 (m, 2H), 3.56-3.70 (m, 2H), 3.39 (m, 2H), 1.18 (d, 12H,j=5.4). ³¹P NMR (162 MHz, CDCl₃): δ 149.1.

(2-Benzyloxy-[(2R,3R)-3-(4-methoxy-benzyloxymethyl)-1,4-dioxa-spiro[4.4]non-2-ylmethoxy]-phosphoryloxy)ethyl)-carbamicacid benzyl ester 5. A solution of the monoprotected alcohol 3 b (486mg, 1.58 mmol, 1 equiv.) and tetrazole (331 mg, 473 mmol, 3 equiv.) indry CH₂Cl₂ (5 mL) was stirred under N₂ for 5 min at rt. Phosphoramidite4 (886 mg, 2.05 mmol, 1.3 equiv.) in dry CH₂Cl₂ (5 mL) was added viacanula, and the mixture was stirred for 2 h (until all starting materialwas consumed). After cooling to 40° C., mCPBA (1.360 g, 6 mmol, 3equiv., 60%) in CH₂Cl₂ (5 mL) was added and the reaction mixture wasstirred for 5 min. The cold bath was then removed and stirring wascontinued for an additional 1 h. The reaction was diluted with CH₂Cl₂and poured into satd. NaHCO₃, and after 20 min of vigorous stirringextracted 3 times with CH₂Cl₂. Combined organic phases were dried(MgSO₄), concentrated in vacuo, and the crude product was purified onSiO₂ (hexane:acetone 3:2) to give 1.033 g (1.57 mmol, 99%) of compound 5as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.28-7.38 (m, 10H),7-18-7-24 (m, 2H), 6.82-6.88 (m, 2H), 5.44-5.54 (m, 1H), 5.00-5.12 (m,4H), 4.42-4.51 (m, 2H), 3.99-4.17 (m, 4H), 3.89-3.99 (m, 2H), 3.76 (s,3H), 3.56 (dd, 1H, J₁=3.6, J₂=7.5), 3.43-3.50 (m, 1H), 3.33-3.41 (m,2H), 1.70-1.84 (m, 4H), 1.54-1.70 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) □159.30, 156.34, 136.47, 135.65, 135.58, 129.92, 129.79, 129.35, 128.68,128.63, 128.48, 128.35, 128.07, 128.03, 119.95, 113.80, 77.24, 76.00,73.18, 69.95, 69.53, 67.46, 66.94, 66.71, 55.21, 53.47, 41.30, 37.26,37.20, 23.49, 23.42; ³¹P NMR (162 MHz, CDCl₃): δ 2.23; IR: 3316, 2956,1722, 1514, 1250, 1023. Anal. Calcd for C₃₄H₄₂NO₁₀P: C, 62.28; H, 6.46;N, 2.14. Found: C, 62.38; H, 6.29; N, 2.18.

(2-{Benzyloxy-[(2R,3R)-2,3-dihydroxy-4-(4-methoxy-benzyloxy)-butoxy]-phosphoryloxy}-ethyl)-carbamicacid benzyl ester 6. To a solution of 5 (2.161 g, 3.3 mmol) in dry THF(100 mL) was added 100 mL of 1 M HCl. The mixture was stirred at rt for3 h and then satd. aq. NaHCO₃ was added. The mixture was transferred toa separatory funnel and extracted with CH₂Cl₂. The crude product waspurified on SiO₂ (toluene:acetone 1:4) to give 1.267 g (2.15 mmol, 65%)of the diol 6 as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.25-7-35(m, 10H), 7.17-7.22 (m, 2H), 6.81-6.86 (m, 2H), 5.64-5.75 (m, 1H),4.95-5.10 (m, 4H), 4.35-4.46 (m, 2H), 3.95-4.12 (m, 4H), 3.78-3.86 (m,1H), 3.65-3.78 (m⁻¹H), 3.75(s, 3H), 3.45-3.55 (m, 2H), 3.30-3.40 (m,2H); ¹³C NMR (100 MHz, CDCl₃) □ 159.31, 156.51, 136.45, 135.50, 129.77,129.42, 128.73, 128.64, 128.48, 128.06, 113.83, 73.14, 71.24, 70.50,69.65, 69.25, 68.90, 66.95, 66.73, 55.22, 41.24; ³¹P NMR (162 MHz,CDCl₃) δ 0.52; IR: 3349, 2954, 1719, 1612, 1513, 1456, 1248, 1023, 821,739, 698. Anal. Calcd for C₂₉H₃₆NO₁₀P: C, 59.08; H, 6.15; N, 2.38; 0,27.14; P, 5.25. Found: C, 58.96; H, 6.27; N, 2.44, P, 5.44.

Hexadecanoic acid{(2R,3R)-3-[benzyloxy-(2-benzyloxycarbonylamino-ethoxy)-phosphoryloxy]-2-hexadecanoyloxy-1-(4-methoxy-benzyloxymethyl)}-propylester 7. DCC (1.377 g, 6.67 mmol, 3 equiv.), and dimethylaminopyridine(DMAP) (299 mg, 2.45 mmol, 1.1 equiv.) were added in one portion to asolution of 6 (1.311 g, 2.22 mmol, 1 equiv.) and hexadecanoic acid(1.901 g, 6.67 mmol, 3 equiv.) in dry CH₂Cl₂. After stirring for 18 h,the reaction mixture was concentrated in vacuo and purified on SiO₂(hexane:acetone 4:1) to give 1.68 g (1.58 mmol, 71%) of product 7 as awaxy solid, mp 60° C. ¹H NMR (400 MHz, CDCl₃) □ 7.26-7.40 (m, 10H),7.18-7.24 (m, 2H), 6.82-6.88 (m, 2H), 5.08 (s, 2H), 4.95-5.06 (m, 2H),4.32-4.46 (m, 2H), 3.88-4.24 (m, 4H), 3.76 (s, 3H), 3.43-3.60 (m, 2H),3.34-3.43 (m, 2H), 2.21-2.32 (m, 4H), 1.50-1.64 (m, 4H), 1.25 (bs, 48H),0.88 (t, 6H, J=5.3); ¹³C NMR (100 MHz, CDCl₃) δ 172.90, 172.77, 159.35,129.41, 128.73, 128.65, 128.47, 128.08, 128.01, 113.80, 72.97, 70.04,69.97, 69.74, 69.66, 69.62, 67.45, 67.02, 66.71, 65.65, 55.18, 41.30,34.15, 31.94, 29.72, 29.67, 29.51, 29.38, 29.31, 29.15, 24.92, 24.86,22.70, 14.12; ³¹P NMR (162 MHz, CDCl₃) δ 0.26, 0.14. IR: 2924, 2853,1741, 1612, 1514, 1456, 1249, 1154, 1112, 1035, 736, 697. Anal. Calcdfor C₆₁H₉₆NO₂P: C, 68.70; H, 9.07; N, 1.31. Found: C, 68.87; H, 8.81; N,1.32.

Hexadecanoic acid{(2R,3R)-3-benzyloxy-(2-benzyloxycarbonylamino-ethoxy)-phosphoryloxy]-2-hexadecanoyloxy-1-hydroxymethyl}-propylester 8. To a solution of 7 (234 mg, 0.23 mmol, 1 equiv.) in CH₂Cl₂ (23mL) was added water (0.23 mL) followed by DDQ (103 mg, 0.46 mmol, 2equiv.). When TLC indicated that the reaction was complete, the mixturewas transferred to separatory funnel and washed with 5% Na₂SO₃ and satd.NaHCO₃ (2×). The aqueous phases were back-extracted once with CH₂Cl₂,and the combined organic phases were dried (MgSO₄), concentrated invacuo, and product was purified on SiO₂ (hexane:acetone 4:1) to give 125mg (0.13 mmol, 60%) of product 8 as a waxy solid, mp ˜60° C. ¹H NMR (400MHz, CDCl₃) δ 7.20-7.40 (m, 10H), 5.0-5.16 (m, 6H), 3.92-4.23 (m, 4H),3.53-3.72 (m, 2H), 3.30-3.45 (m, 2H), 2.80-2.94 (m, 1H), 2.27-2.33 (m,4H), 1.54-1.64 (m, 4H), 1.25 (bs, 48H), 0.88 (t, 6H, J=5.3); ¹³C NMR(100 MHz, CDCl₃) δ 173.44, 173.16, 156.46, 136.45, 135.46, 128.81,128.70, 128.51, 128.13, 128.06, 71.64, 70.05, 69.81, 67.08, 66.80,65.79, 60.70, 41.30, 34.15, 31.94, 29.73, 29.68, 29.52, 29.38, 29.31,29.16, 24.90, 22.70, 14.13; ³¹P NMR (162 MHz, CDCl₃) □ 1.68, 1.61; IR:2917, 2850, 1739, 1467, 1263, 1017. Anal. Calcd for C₅₃H₈₈NO₁₁P: C,67.27; H, 9.37; N, 1.48. Found: C, 67.32; H, 9.49; N, 1.50.

Hexadecanoic acid{(2R,3R)-1-[benzyloxy-(2-benzyloxycarbonylamino-ethoxy)-phosphoryloxymethyl]-3-(benzyloxy-diisopropylamino-phosphanyloxy)-2-hexadecanoyloxy}-propylester 9. To a solution of benzyltetraisopropylphosphordiamidite (1.164g, 3.44 mmol, 1.5 equiv.) and tetrazole (80 mg, 1.15 mmol, 0.5 equiv.)in dry CH₂Cl₂ a solution of ester 8 (2.169 g, 2.30 mmol, 1 equiv.) inCH₂Cl₂ was added via canula. After 2 h, the reaction was concentrated invacuo and the residue was purified on SiO₂ (ethyl acetate:toluene 4:1containing 5% EtN) to give 2.190 g (1.85 mmol, 81%) of phosphoramidite9. ¹H NMR (400 MHz, CDCl₃) δ 7.20-7.40 (m, 10H), 5.0-5.16 (m, 6H),3.92-4.23 (m, 4H), 3.53-3.72 (m, 2H), 3.30-3.45 (m, 2H), 2.80-2.94 (m,1H), 2.27-2.33 (m, 4H), 1.54-1.64 (m, 4H), 1.25 (bs, 48H), 0.88 (t, 6H,V-5.3). ¹³C NMR (100 MHz, CDCl₃) δ 172.91, 172.75, 139.18, 135.58,135.52, 128.74, 128.67, 128.49, 128.31, 128.26, 128.09, 128.02, 127.36,127.29, 126.96, 126.93, 70.59, 69.72, 67.01, 66.72, 65.70, 65.37, 65.20,61.69, 43.10, 34.18, 31.95, 29.73, 29.68, 29.52, 29.38, 29.33, 29.17,24.88, 24.58, 22.70, 14.13; ³¹P NMR (162 MHz, CDCl₃) δ 150.18, 149.79,0.32, 0.22.

Hexadecanoic acid1-[benzyloxy-2-benzyloxycarbonylamino-ethoxy)-phosphoryloxymethyl]-2-hexadecanoyloxy-3[-benzyloxy-phosphoryloxy-3-benzyloxy-2,6-bis(benzyloxymethoxy)-4,5-bis-(bis-benzyloxy-phosphoryloxy)-D-myo-inositol]-propylester 10. To a solution of 4,5-head group (4,5-HG, 250 mg, 0.24 mmol, 1equiv.) and tetrazole (51 mg, 0.73 mmol, 3 equiv.) in dry CH₂Cl₂ wasadded a solution of phosphoramidite 9 (345 mg, 0.29 mmol, 1.2 equiv.) inCH₂Cl₂. The mixture was stirred at rt for about 3 h, cooled to −40° C.and mCPBA (210 mg, 0.73 mmol, 3 equiv., 60%) was added. After 15 min,the cold bath was removed and stirring was continued for additional 1 h.The reaction mixture was diluted with CH₂Cl₂ and poured into a solutionof 5% Na₂SO₃ and satd. NaHCO₃. The mixture was extracted 3 times withCH₂Cl₂ and combined organic fractions were dried over MgSO₄,concentrated in vacuo, and purified on SiO₂ (hexane:acetone 3:2) to give306 mg (0.14 mmol, 59%) of product 10 as a colorless oil. ¹H NMR (400MHz, CDCl₃) □ 7.00-7.35 (m, 50H), 5.55-5.95 (m, 1H), 5.15-5.35 (m, 2H),4.40-5.15 (m, 27H), 4.20-4.40 (m, 2H), 3.90-4.20 (m, 6H), 3.45-3.60 (m,1H), 3.25-3.45 (m, 2H), 2.10-2.30 (m, 4H), 1.40-1.60 (m, 4H), 1.05-1.35(m, 48H), 0.88 (t, 6H, J=5.3); ¹³C NMR (100 MHz, CDCl₃) δ 172.59,138.09, 137.71, 137.25, 136.87, 136.56, 136.23, 136.17, 136.10, 136.06,135.99, 135.90, 135.82, 135.51, 135.54, 128.65, 128.44, 128.34, 128.26,128.02, 127.84, 127.75, 127.42, 96.64, 95.46, 78.99, 77.61, 77.34,76.93, 76.60, 74.89, 72.79, 72.02, 70.45, 69.90, 69.70, 69.44, 69.12,66.97, 66.62, 65.20, 41.29, 33.97, 31.92, 29.70, 29.67, 29.66, 29.51,29.35, 29.29, 29.13, 24.78, 22.68, 14.12; ³¹P NMR (162 MHz, CDCl₃) δ0.35, −0.15, −0.56 (ratio 1:2:1). IR: 2924, 2853, 1743, 1455, 1273,1022. Anal. Calcd for Cl₁₇H₁₅₃NO₂₇P₄: C, 65.99; H, 7.24; N, 0.66; P,5.82. Found: C, 65.75; H, 7.24; N, 0.73; P, 6.12.

1-[(2R,3R)-4-(2-Aminoethoxyphosphoryloxy)-2,3-di-O-palmitoylbutoxyphosphoryloxy]-4,5-myo-bisphosphate11. To a solution of compound 10 (193.4 mg, 0.091 mmol) in a mixture ofTHF/water (4:1, v/v, 50 mL) was added 10% palladium on charcoal (387mg). The mixture was shaken for 18 h at rt under 60 psi of H₂. Thecatalyst was removed by filtration and solvent was removed in vacuo. Thecrude product was redissolved in water and stirred for 3 h with Dowex50×-100 resin (Na⁺ form). The resin was removed by filtration and thefiltrate was lyophilized to give 77.3 mg (0.062 mmol, 62%) as the sodiumsalt. The dried crude product was used for coupling with activated(N-hydroxysuccinimidyl, or “NHS”) esters as described below. ¹H NMR (400MHz, D₂O): □5.15-5.30 (m, 2H), 4.10-4.30 (m, 2H), 3.85-4.10 (m, 7H),3.75-3.85 (m, 1H), 3.6-3.7 (m, 1H), 3.4-3.5 (m, 1H), 3.18 (bs, 2H),2.1-2.5 (m, 4H), 1.4-1.6 (bs, 4H), 1.18 (bs, 48H), 0.76 (bs, 6H). ³¹PNMR (162 MHz, D₂O): δ 2.36, 1.80, 1.09, 0.56 (ratio 1:1:1:1), MS MALDI(free acid): 146 (M+Na), 950 (M+3Na—C₁₅H₃₁CO), 928 (M+2Na—C₁₅H₃₁CO), 906(M+Na—C₁₅H₃₁CO), 884 (M—C₁₅H₃₁CO).

General procedure for coupling with NHS esters. To a solution ofcompound 11 (˜10 μmol, 1 equiv.) in 0.5 M TEAB (0.5 mL, pH 7.5) wasadded a solution of appropriate NHS ester (˜12 □mol, 1.2 equiv.) (threeof which were obtained from Molecular Probes, Inc.) in 0.5 mL ofdimethylformamide (DMF(was added. PROXYL-SE was prepared as described inRauch, M.; Ferguson, C.; Prestwich, G. D.; Cafiso, D. J. Biol. Chem.2002. The mixture was stirred at rt for 18 h, and solvents were thenremoved in vacuo. The residue was washed 4 times with acetone and thenpurified on DEAE-cellulose column with a step gradient of TEAB (0 to 2M). The desired fractions were pooled, lyophilized, converted by ionexchange into a sodium salt, and lyophilized again.

Biotin derivative 12 a. Reaction of 11 (9.4 mg, 7.5 mmol) with Biotin-X,SE (4.4 mg, 9.7 mmol) yielded 6.4 mg (4 mmol, 53%) of 12 a. ¹H NMR (400MHz, D₂O) δ 5.20-5.40 (m, 4H), 4.50-4.60 (m, 1H), 4.30-4.45 (m, 1H),3.70-4.45 (m, 9H), 3.60-3.70 (m, 1H), 3.30-3.45 (m, 1H), 3.05-3.20 (m,4H), 2.65-2.95 (m, 2H), 2.00-2.50 (m, 8H), 1.40-1.80 (m, 12H), 0.90-1.40(m, 52H), 0.70-0.90 (m, 6H); ³¹P NMR (162 MHz, D₂O δ 3.22, 2.32, 1.34,0.41 (ratio 1:1:1:1). MS MALDI (free acid): 1528 (M+3Na), 1506 (M+2Na),1484 (M+Na), 1462 (M−H), 1223 (M−H—C₁₅H₃₁CO), 1122 (M−H-biotin). HRMALDI: C₆₀H₁₁₃N₄O₂₆P₄S [M−H]⁻ calcd: 1461.60388, found: 1461.60491.

NBD Derivative 12 b. Reaction of 11 (9.9 mg, 7.9 mmol) with NBD-X, SE(4.0 mg, 10.2 mmol) afforded 7.7 mg (5 mmol, 64%) of 12 b. ¹H NMR (400MHz, D₂O): δ 8.10-8.30 (m, 1H), 6.00-6.20 (m, 1H), 5.10-5.30 (m, 2H),3.65-4.20 (m, 12H), 3.20-3.65 (m, 5H), 2.95-3.25 (m, 4H), 2.20-2.40 (m,2H), 2.15-2.25 (m, 4H), 1.65-1.80 (m, 2H), 1.50-1.65 (m, 2H), 1.30-1.50(m, 4H), 0.80-1.30 (m, 50H), 0.50-0.80 (m, 6H); ³¹P NMR (162 MHz, D₂O):δ 4.03, 2.92, 1.37, 0.47 (ratio 1:1:1:1). MS MALDI (free acid): 1161(M−C₁₅H₃₁CO).

Fluorescein derivative 12 c. Reaction of 11 (9.5 mg, 7.6 mmol) with6-FAM-SE (4.7 mg, 9.8 mmol) yielded 9.1 mg (5.6 mmol, 74%) of 12 c. ¹HNMR (400 MHz, D₂O): δ 7.30-7.50 (m, 8H), 5.20-5.40 (m, 2H), 3.10-4.30(m, 14H), 2.20-2.40 (m, 4H), 1.40-1.60 (m, 4H), 0.90-1.40 (m, 48H),0.60-0.90 (m, 6H); ³¹P NMR (162 MHz, D₂O): δ 4.94, 4.33, 1.21, 0.65(ratio 1:1:1:1). MS MALDI (free acid): 1243 (M−C₁₅H₃₁CO), 1123(M-fluorescein), 884 (M−C₁₅H₃,CO-fluorescein), 841(M−C₁₅H₃₁CO-fluorescein-aminoethyl).

PROXYL derivative 12 d. Reaction of 11 (9.8 mg, 7.8 mmol) with PROXYL-SE(2.9 mg, 10.1 mmol) afforded 6.1 mg (4.3 mmol, 55%) of 12 d. MS MALDI(free acid): 1292 (M), 1123 (M-proxyl), 1053 (M−C₁₅H₃₁CO), 884(M−C₁₅H₃₁CO-proxyl). HR MALDI: C₅₃H₁₀₃N₂O₂₅P₄ [M]⁻ calcd: 1291.57950;found: 1291.57679.

The resulting exemplary Pea-PIP_(n), 2,3-diacylthreitol-basedPea-Ptdlns(4,5)P₂, (“Pea-PIP2”) possesses a phosphatidylethanolamine(Pea) head group at the 1-position and a phosphatidylinositol4,5-bisphosphate (Ptdlns(4,5)P₂) head group at the 4-position. Reporters(biotin, fluorophores, spin label) were covalently attached to the freeamino group of the Pea, such that these reporters were targeted to thelipid-water interface. See FIG. 5. The diacyl moieties allowincorporation of Pea-PIP₂ into a lipid bilayer, while the Ptdlns(4,5)P₂moiety in the aqueous layer is specifically recognized byPtdlns(4,5)P₂-specific binding proteins. Reaction of the free Pea aminogroup of with four N-hydroxysuccinimidyl (NHS) esters afforded thecorresponding biotinylated derivative C-2 a, the fluorescentN-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD) and 6-carboxyfluoresceinderivatives C-2 b and C-2 c, and the spin-labeled3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PROXYL) derivative C-2d. Preliminary biological results are described below for exemplarybiotinylated and fluorescent derivatives.

Example 2 Synthesis of Pea-PIP_(n)s Having Eight Different NaturallyOccurring Phosphoinositide Head Groups

Synthetic representatives of eight naturally occurring phosphoinositidehead groups have been incorporated into Pea-PIP_(n)s of the presentinvention. The synthetic method for producing these was as describedabove for Pea-PI(4,5)P₂ and its reporter derivatives. The syntheticstrategy for producing all eight of these Pea-PIP_(n)s is described inTable 1 and FIG. 2. The head groups used in this Example to producePea-PIP_(n)s of the invention are, Pi, PI(3)p, PI(4)P, PI(5)P,PI(3,4)P₂, PI(3,5)P₂, PI(4,5)P₂, PI(3,4,5)P₃. TABLE 1 HeadgroupProtected Deprotected PI, R₁, R₁, R₃═Bn R⁴, R⁵, R⁶═H PI(3)p R₁, R₂═Bn,R₃═PO₃Bn₂ R¹, R²═H, R³═PO₃H₂ PI(4)P R₁, R₃═Bn, R₂═PO₃Bn₂ R¹, R³═H,R²═PO₃H₂ PI(5)P R₂, R₃═Bn, R₁═PO₃Bn₂ R², R³═H, R¹═PO₃H₂ PI(3,4)P₂ R₁═Bn,R₂, R₃═PO₃Bn₂ R¹═H, R³═PO₃H₂ PI(3,5)P₂ R₂═Bn, R₁, R₃═PO₃Bn₂ R²═H, R¹,R³═PO₃H₂ PI(4,5)P₂ R₃═Bn, R₁, R₂═PO₃Bn₂ R³═H, R¹, R²═PO₃H₂ PI(3,4,5)P₃R₁, R₂, R₃═PO₃Bn₂ R¹, R², R³═PO₃H₂

Example 3 Synthesis of Linker-Modified Derivatives of Pea-PIP_(n)s

-   -   a. Hydrophilic linker-modified Pea-PIP_(n) analogs. Preliminary        data from immobilization of Pea-PIP_(n)s and a hydrophilic        linker-modified analog to functionalized surfaces suggests that        increasing the distance between the PIP_(n) head group and the        probe moiety may increase ligand recognition. A hydrophilic        linker-modified Pea-PIP_(n) derivative was synthesized in order        to increase the distance between the PIP_(n) head group and the        probe moiety (see FIG. 6). In a first example of linker        extension, amino-PEG-amide linker-extended Pea-PI(4,5)P₂ was        prepared from the parent Pea-PI(4,5)P₂ by coupling the primary        amine with the NHS ester of a 16-atom linker purchased as a Fmoc        protected activated ester (available commercially, for example        from Quanta Biodesign, Inc.). This derivative was examined for        binding to the PLC_(δ) PH domain using AlphaScreen® (available        from PerkinEimer Life Sciences), PIP Arrays™ (available from        Echelon Biosciences, Inc.), and immobilized on NHS-activated        plates or beads. Increased binding to a pleckstrin homology        domain was observed with the addition of the hydrophilic linker.    -   b. Additional PEG linker Pea-PIP_(n) analogs. Various lengths of        poly(ethylene glycol)-based linkers can be used for preparation        of alternative linker-modified Pea-PIP_(n)s. In a particular        embodiment, PEG linkers were prepared from commercially        available di-, tri-, tetra-, and/or penta(ethylene glycols). In        the first step, the linkers were transformed into mono        p-toluenesulfonates (Bauer, H., Sber, F., Petry, C., Knorr, A.,        Stadler, C., and Staab, H. A. (2001) European Journal of Organic        Chemistry, 3255-3278), then converted into the        oligo-PEG-{overscore (ω)}-aminoalcohols (Nelissen, H. F. M.,        Venema, F., Uittenbogaard, R. M., Feiters, M. C., and        Nolte, R. J. M. (1997) Journal of the Chemical Society, Perkin        Transactions 2: Physical Organic Chemistry, 2045-2053;        Bramson, H. N., Corona, J., Davis, S. T., Dickerson, S. H.,        Edelstein, M., Frye, S. V., Gampe, R. T., Jr., Harris, P. A,        Hassell, A., Holmes, W. D., Hunter, R. N., Lackey, K. E.,        Lovejoy, B., Luzzio, M. J., Montana, V., Rocque, W. J., Rusnak,        D., Shewchuk, L., Veal, J. M., Walker, D. H., and        Kuyper, L. F. (2001) Journal of Medicinal Chemistry 44,        4339-4358), and finally protected with CbzCl in        dichloromethane/triethylamine (Roy, B. C., and Mallik, S. (1999)        Journal of Organic Chemistry 64, 2969-2974). In this Example,        linker extensions including 2-(2-Amino-ethoxy)-ethanol and        2-(2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy)-ethanol were introduced        at an early stage of Pea-PI(4,5)P₂ synthesis, specifically        during the preparation of the phosphoramidites. FIG. 7        illustrates the synthetic strategy for preparation of four        oligo-PEG linkers. Relatively short PEG linkers are chosen to        avoid floppiness, foldback, and heterogeneity often experienced        with MW 700 or 1500 or 3400 PEG derivatives. In certain        embodiments of the present invention, PEG linkers can be of any        size. In particular embodiments, such PEG linkers may range from        MW 88-3400 (44-20,000 Daltons). Suitably protected aminoalcohols        are next converted into the corresponding phosphoramidites and        coupled with specifically protected threitol as shown in FIG. 8.        Following coupling, oxidation, and deprotection as previously        described and shown in FIG. 2 and Table 1, three oligoethylene        glycol phosphoramidite modified Pea-PIP, analogs were obtained.

Example 4 Pea-PIP_(n)s are Substrates for Lipid Phosphatase or Kinase

This Example demonstrates further that Pea-PIP_(n)s of the presentinvention are effective substrates for lipid phosphatases and kinases.In this Example, Pea-PIP_(n)s-were shown to be effective and specificsubstrates for recombinantly expressed and purified recombinant humanPTEN/MMAC1 (acronyms for Phosphatase and Tensin homolog and mutated inmultiple advanced cancers). PTEN/MMAC1 is a lipid phosphatase thatremoves the 3′ phosphate from PI(3,4,5)P₃ to produce PI(4,5)P₂.According to this Example, purified Glutathione S-Transferase(GST)-tagged, PTEN (0.24 mg/ml in 50% glycerol 50% elution buffer) wasadded to 125 pmol Pea-PI(3,4,5)P₃ in 50 ul reaction buffer (100 mM TRIS,pH 8.0, 10 mM DTT) (chemicals from Sigma-Aldrich Life Science, St.Louis, Mo.). The reactions were incubated for one hour at 37° C., anddetected using an AlphaScreen™ assay. A standard curve of the productwas generated by adding serial dilutions of Pea-PI(4,5)P₂ as thecompetitor to separate wells in the same plate. Results of this assayshowed that two separate enzyme preparations of PTEN were active againstPea-PI(3,4,5)P₃. Using the standard curve, it was estimated that 48% ofthe Pea-PI(3,4,5)P₃ substrate was converted to Pea-PI(4,5)P₂ during thecourse of the reaction. These results show that members of this uniqueand novel class of phosphoinositide analogs are acted on as substratesin assay platforms for lipid kinases and phosphatases. Although thisExample uses PTEN and detection of Pea-PI(4,5)P2; however, it isenvisioned that this system extends to any combination of Pea-PIPnheadgroup detection and any phosphoinositide kinase or phosphatase.

Example 5 Immobilized Pea-PIP_(n)s are substrates for Lipid RecognizingProteins

In this Example, immobilized Pea-PIP_(n)s are readily and specificallyrecognized by lipid recognition proteins. According to this Example, sixdifferent quantities (from 200 pmol to 6 pmol) of Ptdlns(3,4)P₂,Ptdlns(3,5)P₂, Ptdlns(4,5)P₂, Pea-PI(4,5)P₂,Ptdlns(3,4,5)P₃;Pea-PI(3,4,5P)₃ PI and PE were spotted onto nitrocellulose, and bindingof Glutathione S-Transferase (GST)-PLC δ₁-PH and GST-General Receptorfor Phosphoinositides (Grp1)-PH constructs was examined by aprotein-overlay technique (described in Dowler, S., Kular, G., andAlessi, D. R. (2002) Sci STKE 2002, PL6) (recombinant GST tagged PLCδ₁-PH and Grp1-PH domain proteins used in this Example were expressed inE. coli then purified using glutathione affinity resin (AmershamBiosciences, Piscataway, N.J.). The results of this binding assay showthat neither lipid-recognizing protein (LRP) recognizes the PE or PIcontrol lipids, nor do they recognize the non-cognate phosphoinositides,but both LRPs showed dose-dependent recognition of the correctimmobilized phosphoinositide. Significantly, both Pea-PI(4,5)P₂ andPea-PI(3,4,5)P₃ are able to bind the correct protein at lowerconcentrations than the corresponding diC₁₆ phosphoinositides,demonstrating that the Pea-PIP_(n)s have improved LRP bindingcapabilities compared to the current standard PIP_(n) lipids.

Example 6 Biotinylated Pea-PIP_(n)s are Substrates for Lipid RecognizingProteins

This example demonstrates that a phosphoinositide head-group specificlipid-recognizing protein (LRP), namely GST-Phospholipase Cδ₁ (PLC δ₁)PH domain, will easily bind to the biotinylated derivative ofPea-PI(4,5)P₂, even when bound to a surface.

According to this example, biotinylated lipid Pea-PI(4,5)P₂ was bound toa streptavidin-coated donor bead and the GST-tagged PLC δ₁—PH domain wasattached to an anti-GST-coated acceptor bead. A luminescent signalquantitatively reported the interaction between the biotinylated lipidand the binding protein. Further, in the absence of a lipid or aspecific binding protein, no signal was seen. In the presence of 0.1pmol/well of specific binding protein, biotinylated Pea-PI(4,5)P₂ showeda dose-dependent increase in luminescent signal up to 1 pmol per well.

In this experiment, a bioluminescence assay (AlphaScreen™, Perkin-ElmerLife Sciences, Boston, Mass.) was used to establish biochemicalrelevance of the claimed compounds. Binding of Pea-PI(4,5)P2 to PLC δ₁was determined using recombinant GST tagged PLC 5-PH domain protein thatwas expressed in E. coli, then purified using glutathione affinity resin(Amersham Biosciences, Piscataway, N.J.). Several concentrations ofpurified protein and biotinylated-Pea-PIP2 (all phosphoinositides werefrom Echelon Biosciences Inc, SLC, UT) were combined in a white 384-wellmicroplate (Optiplate™, Packard Bioscience, Meriden, Conn.).Streptavidin donor and Anti-GST acceptor beads (Perkin-Elmer lifesciences) were then added in a light protected area, so that the finalbead concentration is 5 μg/mL in 25 μL final reaction volume (alldilutions in AlphaScreen assay buffer, Tris-Buffered Saline pH 7.5, 0.1%Tween-20, 0.1% Bovine Serum Albumin). The plate was protected from lightand incubated for 2 hours at room temperature before reading with theAlphaScreen mode of a Fusion instrument (Perkin-Elmer life sciences).

In addition, competitive binding assays were conducted in which theGST-PLC δ₁-PH protein was pre-incubated for 30 min with 10 nM to 10 fMof unlabeled diC₄ Ptdlns(4,5)P₂, Ptdlns(3,4,5)P₃, or Pea-PI(4,5)P₂ priorto addition of the other reagents, using biotinylated Pea-PI(4,5)P₂ asthe probe lipid. Over 100-fold selectivity was observed for displacementof the GST-PLC δ₁-PH from binding to Pea-PI(4,5)P₂ by the di-C₄Ptdlns(4,5)P₂, relative to di-C₄ Ptdlns(3,4,5)P₃. A further increase inbinding to the PH domain of GST-PLC 61 was observed using thenon-biotinylated version of Pea-PI(4,5)P₂.

Example 7 Specific Recognition and Binding by Anti-PIP, Antibody toPea-PIP_(n)s Covalently Bound to a Surface

The ability of lipid recognition proteins to specifically recognize andbind to Pea-PIP_(n)s was tested by coupling Pea-PIP_(n)s (as well asamino PIPs with the same headgroups as controls) to polystyrenemicrotiter plates. Briefly, 50 pmol per well of amino PI(4,5)P₂ (PIP2),amino PI(3,4,5)P₃ (PIP3), Pea-PI(4,5)P₂ (Pea-PIP2), and Pea-PI(3,4,5)P₃(Pea-PIP3) lipids in 100 μL PBS were coupled via their primary aminefunctional groups to triplicate wells of a Maleic-Anhydride activated96-well plate (Pierce, Rockford, Ill.). Underivatized groups werereacted with 200 μL Tris-Glycine-SDS overnight before blocking with 200μL 0.02% Ovalbumin in TBS. The plate was then incubated with anti-PIP₃antibody (for example, NN111.1.1, MBL International, Watertown, Mass.)for one hour at room temperature on an orbital shaker. The plate waswashed 3-5 times with 200 μL/well of TBS containing 0.1% Tween-20.Specific binding was subsequently visualized by incubating withanti-mouse-HRP (horseradish peroxidase) secondary antibody (Sigma, StLouis, Mo.), washing as before, then adding tetramethylbenzadine (TMB)developing reagent (Sigma) and reading the absorbance at 450 nm in aplate reader. Anti-PIP3 antibody correctly and specifically bound toPea-PI(3,4,5)P₃, demonstrating that, similar to the control lipids,Pea-PIP₃ (but not Pea-PIP₂) was recognized by the specific antibody. Inaddition, Pea-PIP₃ gave an increased signal compared to ω-amino alkanoylPIP₃. These results show that immobilizing Pea-PIPs either bynon-specific adsorption or covalent coupling allows the structuralfeatures necessary for recognition by antibodies and lipid recognizingproteins (namely, the headgroup and two fatty-acyl chains) to be betterpositioned than in traditional PIPs.

Example 8 Intracellular uptake of Pea-PIPs

Pea-PIP_(n)s can be delivered into living cells using a commerciallyavailable system (Echelon Bioscience Shuttle PIP™ system (Salt LakeCity, Utah)). A fluorescent Pea-PI(4,5)P₂ analog was delivered to cellsusing the Shuttle PIP™ technology. This method has previously been usedto deliver fluorescent PtdlnsP_(n) analogs, Ptdlns(3,4)P₂ for ProteinKinase B (Akt) activation, and Ptdlns(3,4,5)P₃ to induce cell migration.3T3L1 preadipocyte cells were seeded onto an 8-well cover-glass chamberslide in complete media. After 24 hrs the cells were approximately 60%confluent and the media was replaced with 100 μL serum free media for 45minutes before adding a mixture of fluorescent Pea-PI(4,5)P₂-NBD andHistone H1 carrier premixed and incubated at room temperature for 10minutes, final concentration of Pea-PI(4,5)P₂-NBD was 12.5 μM; andHistone carrier, 2.5 μM). After 30 minutes, the cells were imaged with aBio-Rad confocal microscope at 300× magnification. Results of this assayshowed that Pea-PI(4,5)P₂-NBD localized to intracellular compartmentswith bright staining associated in specific regions of the plasmamembrane. This pattern of intracellular localization positionsPea-PI(4,5)P₂ correctly in the cell to substitute for endogenousPtdlns(4,5)P₂ in signaling pathways and cell-based assays.

Example 9 PeaPIPns can substitute for synthetic PIPns on PIP Strip*Products

Pea-PIP_(n)s can substitute for synthetic PIP_(n)s on PIP Strip*products (Echelon, Salt Lake City, Utah). Briefly, 100, 50, 25, 12.5,6.25, and 3.125 pmol of lipids in organic solvent were spotted onto PVDFmembrane (Amersham, Boston, Mass.) and allowed to dry before blockingthe membrane with 0.1% Ovalbumin in TBS. The membranes were thenincubated with GST-PH domain proteins (LRPs) specific for PI(4,5)P2 orPI(3,4,5)P₃ for one hour at room temperature on an orbital shaker.Binding was visualized by subsequent incubations of anti-GST andanti-Mouse-HRP secondary antibodies followed by ECL (enhancedchemiluminescence) detection and exposure to photographic film. ThePea-PIPn's demonstrated the correct specificity for LRP binding and weresuperior to regular synthetic PIPs by immobilizing both PI(4,5)P₂ andPI(3,4,5)P₃-specific proteins at lower lipid concentrations.

Example 10 Pea-PIPs are capable of Adhesion to Microtiter Plates

Binding specificity of Pea-PIPs was further tested by coupling Pea-PIP2and Pea-PIP3 (as well as amino PIPs with the same headgroups ascontrols) to polystyrene microtiter plates. Briefly, 50 pmol per well ofamino PI(4,5)P₂ (PIP₂), amino PI(3,4,5)P₃ (PIP₃), Pea-PI(4,5)P₂(PEA-PIP2), and Pea-PI(3,4,5)P₃ (PEA-PIP₃) lipids in 100 μL PBS werecoupled via their primary amine functional groups to triplicate wells ofa Maleic-Anhydride activated 96-well plate (Pierce, Rockford, Ill.).Underivatized groups were reacted with 200 uL Tris-Glycine-SDS overnightbefore blocking with 200 uL 0.02% Ovalbumin in TBS. The plate was thenincubated with anti-PIP₃ antibody (for example, NN111.1, MBLInternational, Watertown, Mass.) for one hour at room temperature on anorbital shaker. The plate was washed 3-5 times with 200 μL/well of TBScontaining 0.1% Tween-20. Specific binding was subsequently visualizedby incubating with anti-mouse-HRP secondary antibody (Sigma, St Louis,Mo.), washing as before, then adding Tetramethylbenzadine (TMB)developing reagent (Sigma) and reading the absorbance at 450 nm in aplate reader. Similar to the control lipids; Pea-PIP₃ (but not Pea-PIP₂)was recognized by the specific antibody. In addition Pea PIP₃ gave anincreased signal compared to amino PIP₃, similar to the nitrocelluloseexperiment. This Example demonstrates that immobilization of Pea-PIPseither by non-specific adsorption or covalent coupling allows thestructural features necessary for recognition by antibodies and lipidrecognizing proteins (namely, the headgroup and two fatty-acyl chains)to be better positioned than traditional PIPs.

Example 11 Pea-PIPs are Capable of Transfer into Living Cells

Pea-PIP_(n)s can be delivered into living cells using Echelon's ShuttlePIP system (Salt Lake City, Utah). 3T3-L1 fibroblasts in modified DMEMmedia (Gibco BRL, Maryland) were seeded onto an 8-well coverglasschamber slide ((Nalge Nunc International, Naperville, Ill.), 200 μL perchamber in complete media. After 24 hrs the cells were 60% confluent andthe media was replaced with 100 μL serum free media for 45 minutesbefore 2.5 μL of 5 mM fluorescent Pea-PI(4,5)P₂-NBD was added to 6.25 μLof 200 μM Histone H1 (Sigma, St. Louis, Mo.) and incubated at roomtemperature for 10 minutes. Then 16.25 μL serum-free media was added tothe PIP/Histone complex and incubated for an additional 5 minutes beforeit was added to the cells in a final volume of 125 μL and a finalconcentration of 100 μM Pea-PI(4,5)P₂ and 10 μM Histone H1. The cells oncoverslips were imaged with a Bio-Rad confocal microscope at 60×magnification. This procedure was repeated in a separate experiment witha final Pea-PI(4,5)P₂ concentration of 12.5 μM and Histone concentrationof 2.5 μM then visualized at 300× magnification. Pea-PI(4,5)P₂-NBDclearly localized to intracellular compartments with bright stainingassociated in several regions of the plasma membrane. This pattern ofintracellular localization positioned Pea-PI(4,5)P₂ correctly in thecell to substitute for endogenous PI(4,5)P₂ in signaling pathways andcell-based assays.

Example 12 Pea-PIPn's can substitute for synthetic PIPs in lipid kinaseand lipid phosphatase assay development programs

A Phosphoinositide head-group specific lipid-recognizing protein (LRP),namely GST-Phospholipase Cd1 PH domain, will easily bind to thebiotinylated derivative of Pea PI(4,5)P₂. An AlphaScreen technology(Perkin-Elmer Life Sciences, Boston, Mass.) is utilized for ourlipid-protein binding assay in this example.

AlphaScreen (for Amplified Luminescent Proximity Homogenous Assay) is achemiluminescent, bead-based assay performed in white micro-titerplates. When excited by 680 nm laser light, donor beads convert ambientoxygen to a more excited singlet state. When an acceptor bead is inclose proximity to the donor bead (through a biological interaction)singlet oxygen reacts with a thioxene derivative in the acceptor beadgenerating chemiluminescence light of 370 nm wavelength which furtherexcites flourophores on the same acceptor bead emitting light at 520-620nm.

Binding of PEA PI(4,5)P₂ to PLCd1 was determined by expressingrecombinant GST tagged PLC-d1-PH domain protein in E. coli which waspurified using glutathione affinity resin (Amersham Biosciences,Piscataway, N.J.). Several concentrations of purified protein andbiotinylated-PEA PIP₂ (all phosphoinositides were from EchelonBiosciences Inc, SLC, UT) were combined in a white 384-well microplate(Optiplate™, Packard Bioscience, Meriden, Conn.). Streptavidin donor andAnti-GST acceptor beads (Perkin-Elmer life sciences) were then added ina light protected area, so that the final bead concentration is 5 μg/mLin 25 μL final reaction volume (all dilutions in AlphaScreen assaybuffer, Tris-Buffered Saline pH 7.5, 0.1% Tween-20, 0.1% Bovine SerumAlbumin). The plate was protected from light and incubated for 2 hoursat room temperature before reading with the AlphaScreen mode of a Fusioninstrument (Perkin-Elmer life sciences).

A strong binding interaction between biotinylated Pea-PI(4,5)P₂ and thePH domain of PLC-δ-1 was observed with maximum signal at 0.8 pmol/wellbiotinylated Pea-PI(4,5)P₂ and 0.4 pmol/well PLC-δ-1. Half theseconcentrations also produced a strong signal and were used as bindingpartners in the next series of experiments to demonstrate specificity ofPea-PIP binding to PLC-δ 1. These competitive binding experiments weresimilar to the binding experiment described above except that nonbiotinylated lipids are added to each reaction to compete withbiotinylated Pea-PI(4,5)P₂ for binding to PLC-δ 1-PH domain. Forexample, eight concentrations of PI(3,4,5)P₃ diC4, PI(4,5)P₂ diC4, andPea-PI(4,5)P₂ were added as competitors to separate wells in addition to0.2 pmol/well PLC-δ-1-PH domain, 0.4 pmol/well of Pea-PI(4,5)P₂,streptavidin donor, and anti-GST acceptor beads; and incubated aspreviously described. It was determined that Pea-PI(4,5)P₂ was the bestcompetitor with an IC50 value of 140 nM, and the incorrect headgroupPI(3,4,5)P₃ demonstrated the least affinity with an IC50 value of 70 mM.This 500 times better affinity of Pea-PI(4,5)P₂ for PLC d1-PH domaincompared to PI(3,4,5)P₃ is fundamental to the development of a robustassay where one detects the specific PIPn product of a kinase orphosphatase in the presence of excess PIPn substrate.

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the COMPOSITIONS, METHODS and APPARATUS and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A functionalized phosphoinositide polyphosphate comprising a carbonbackbone, a phosphatidylethanolamine head group at carbon position 1,and a PtdlnsP_(n) head group at carbon position
 4. 2. A functionalizedphosphoinositide polyphosphate of claim 1, wherein said carbon backboneis selected from the group consisting of 2,3-diacylthreitol, erythritoland a synthetic module.
 3. A functionalized phosphoinositidepolyphosphate of claim 2, wherein said synthetic module is prepared andcoupled with a PtdlnsP_(n) head group.
 4. A functionalizedphosphoinositide polyphosphate of claim 3, wherein said PtdlnsP_(n) headgroup is selected from the group consisting of (PI, PI(3)_(p), PI(4)P,PI(5)P, PI(4,5)P₂, PI(3,4)P₂, PI(3,5)P₂, PI(3,4,5)P₃).
 5. Afunctionalized phosphoinositide polyphosphate of claim 1, furthercomprising a reporter group.
 6. A functionalized phosphoinositidepolyphosphate of claim 5, wherein the reporter is selected from thegroup consisting of a flourophore, a spin label, biotin, a radio label,a chemiluminescent label, a photophore, a chromophore, a nanogoldparticle and mixtures thereof.
 7. A functonalized phosphoinositidepolyphosphate of claim 6, wherein the flourophore is selected from thegroup consisting of acrylodan, AMCA, BODIPY, Cascade-Blue, CINERF,dansyl, dialkylaminocoumarin, eosin, erythrosine, fluorescein,hydroxycoumarin, NBD, Oregon green, PyMPO, pyrene, rhodamine, RhodolGreen, TMR, Texas Red, and X-Rhodamine.
 8. A functionalizedphosphoinositide polyphosphate of claim 5, wherein the reporter iscovalently attached to a free amino group on thephosphatidylethanolamine head group.
 9. A functionalizedphosphoinositide polyphosphate of claim 8, wherein the reporter isattached to a free amino group by a linker.
 10. A functionalizedphosphoinositide polyphosphate of claim 9, wherein said linker comprisesan oligo-polyethylene glycol linker.
 11. A functonalizedphosphoinositide polyphosphate of claim 9, wherein said linker mayinclude an additional or alternative linker.
 12. A functionalizedphosphoinositide polyphosphate of claim 11, wherein said additional oralternative linker is selected from the group consisting of a diaminolinker, a linker utilizing both phospho- and non-phospho linked spacers,a phosphatidylserine linker and a carboxylic acid linker.
 13. Afunctionalized phosphoinositide polyphosphate of claim 11, wherein saidlinker includes aminoalcohols, heteroatom-containing derivatives orbranched aminoalcohols.
 14. A functionalized phosphoinositidepolyphosphate of claim 13, further comprising a diamino linker.
 15. Afunctionalized phosphoinositide polyphosphate of claim 1 selected fromthe group consisting of Pea-PI, Pea-PI(3)P, Pea-PI(4)P, Pea-PI(5)P,Pea-PI(3,4)P₂, Pea-PI(3,5)P₂, Pea-PI(4,5)P₂ and Pea-PI(3, 4,5)P₃.
 16. Afunctionalized phosphoinositide polyphosphate of claim 1, wherein thecarbon backbone comprises 2 acyl chains having from 2 to 26 carbons. 17.A functionalized phosphoinositide polyphosphate of claim 1, wherein thecarbon backbone comprises an ether chain in the place of one or bothacyl chains.
 18. A functionalized phosphoinositide polyphosphate ofclaim 1, further comprising one or more phosphate groups that have beenchemically modified to stabilize the compound against chemical orenzymatic hydrolysis.
 19. A functionalized phosphoinositidepolyphosphate of claim 1, further comprising a polymerizable group. 20.A method of screening for phosphoinositide-specific binding proteinscomprising (a) contacting a functionalized phosphoinositidepolyphosphate comprising a carbon backbone, a phosphatidylethanolaminehead group at carbon position 1, and a PtdlnsP_(n) head group at carbonposition 4 with a putative phosphoinositide-specific bindingprotein-containing composition; and (b) measuring binding
 21. A methodof claim 20, wherein said functionalized phosphoinositide polyphosphateis attached to a surface.
 22. A method of claim 21, wherein said surfaceis selected from the group consisting of plates, beads, liposomes,nitrocellulose and metals.
 23. A method of claim 21, wherein saidattaching a functionalized phosphoinositide polyphosphate is selectedfrom the group consisting of streptavidin and NHS activation.
 24. Amethod of claim 20, wherein said functionalized phosphoinositidepolyphosphate is selected from the group consisting of Pea-PI,Pea-PI(3)P, Pea-PI(4)P, Pea-PI(5)P, Pea-PI(3,4)P₂, Pea-PI(3,5)P₂,Pea-PI(4,5)P₂ and Pea-PI(3, 4,5)P₃.
 25. A method of claim 20, whereinsaid functionalized phosphoinositide polyphosphate further comprises areporter.
 26. A method of claim 25, wherein said reporter is selectedfrom the group consisting of a flourophore, a spin label, biotin, aradio label, a chemiluminescent label, a photophore, a chromophore, ananogold particle and mixtures thereof.
 27. A method of claim 20,wherein said method comprises an assay selected from the groupconsisting of an in vitro enzyme assay, an in vitro agonist assay, an invitro antagonist assay, a cell-based assay, a lipid kinase activityassay, a protein kinase activity assay, a lipid phosphatase activityassay, a protein phosphatase activity assay, a phospholipase assay and aphosphatase activity assay.
 28. A method of claim 20, wherein saidphosphoinositide-specific binding protein is selected from the groupconsisting of protein kinases, phosphoinositide kinases,phosphoinositide binding proteins, inositol phosphate binding proteins,lipid phosphatases and phospholipases.
 29. A method of identifyingcompositions that affect phosphoinositide recognition or signaling at acell membrane comprising (a) contacting a functionalizedphosphoinositide polyphosphate comprising a carbon backbone, aphosphatidylethanolamine head group at carbon position 1, and aPtdlnsP_(n) head group at carbon position 4 with a composition thatputatively affects phosphoinositide recognition or signaling; and (b)measuring recognition and signaling.
 30. A method of claim 29, whereinsaid composition that putatively affects phosphoinositide recognition orsignaling comprises a compound selected from the group consisting of achemical, a natural product, and a synthetic compound.
 31. A method ofclaim 29, wherein said composition that putatively affectsphosphoinositide recognition or signaling comprises a compound selectedfrom the group consisting of lipid phosphatases and phospholipases. 32.A method of claim 28, wherein said method comprises a cell-based assay.33. A functionalized phosphoinositide polyphosphate comprising Pea-PI,Pea-PI(3)P, Pea-PI (4)P, Pea-PI(5)P, Pea-PI (3, 4)P₂, Pea-PI (3,5)P₂,Pea-PI(4,5)P₂ or Pea-PI(3,4,5)P₃.